Solid-phase synthesis and biochemical evaluation of conformationally constrained analogues of deglycobleomycin A5

Article abstract of DOI:10.1016/j.bmc.2003.08.033

Deglycobleomycin binds to and degrades the self-complementary oligonucleotide d(CGCTAGCG)₂ in a sequence selective fashion. A previous modeling study [J. Am. Chem. Soc. 120, (1998), 7450] had shown that, during binding to double stranded DNA, the conformation of the methylvalerate domain of deglycoBLM approximated that of S-proline. In the belief that an analogue of deglycoBLM structurally constrained to mimic the DNA-bound conformation might exhibit facilitated DNA binding and cleavage, an analogue of deglycoBLM was prepared in which the methylvalerate moiety was replaced by S-proline. This deglycoBLM analogue, as well as the related analogue containing R-proline, was synthesized on a TentaGel resin. Both of the analogues were found to be capable of binding Fe²⁺ and activating O₂ for transfer to styrene. However, both deglycoBLM analogues exhibited diminished abilities to effect the relaxation of supercoiled plasmid DNA, and neither mediated sequence selective DNA cleavage.

Full text of DOI:10.1016/j.bmc.2003.08.033

Bioorganic & Medicinal Chemistry 11 (2003) 5179–5187
Solid-Phase Synthesis and Biochemical Evaluation of
Conformationally Constrained Analogues of Deglycobleomycin A5
Ali Cagir, Zhi-Fu Tao, Steven J. Sucheck and Sidney M. Hecht*
Departments of Chemistry and Biology, University of Virginia, Charlottesville, VA 22901, USA
Received 8 May 2003; accepted 11 August 2003
Abstract—Deglycobleomycin binds to and degrades the self-complementary oligonucleotide d(CGCTAGCG) in a sequence selec-
2
tive fashion. A previous modeling study [J. Am. Chem. Soc. 120, (1998), 7450] had shown that, during binding to double stranded
DNA, the conformation of the methylvalerate domain of deglycoBLM approximated that of S-proline. In the belief that an ana-
logue of deglycoBLM structurally constrained to mimic the DNA-bound conformation might exhibit facilitated DNA binding and
cleavage, an analogue of deglycoBLM was prepared in which the methylvalerate moiety was replaced by S-proline. This degly-
coBLM analogue, as well as the related analogue containing R-proline, was synthesized on a TentaGel resin. Both of the analogues
2
+
were found to be capable of binding Fe and activating O
diminished abilities to effect the relaxation of supercoiled plasmid DNA, and neither mediated sequence selective DNA cleavage.
2
for transfer to styrene. However, both deglycoBLM analogues exhibited
#
2003 Elsevier Ltd. All rights reserved.
Introduction
C-terminal domain of BLM is important for DNA
1
7
binding. The importance of the linker domain, com-
prised of the dipeptide between the DNA binding and
metal binding domains, has been the subject of a num-
ber of recent studies.
The bleomycins (BLMs), originally isolated from a fer-
mentation broth of Streptomyces verticillus by Umezawa
and coworkers, are structurally related, glycopeptide
derived antitumor antibiotics used extensively in the
clinic for the treatment of several types of cancers. It is
believed that the anticancer activity of BLM is due to
the oxidative degradation of DNA
1
2
Based on NMR measurements and molecular dynamics
18ꢀ21
.
calculations of Co BLM–DNA complexes
and the
study of deglycoBLM analogues structurally modified
3
,4
and possibly
5ꢀ7
22ꢀ24
RNA
metal ion such as Fe
cleavage of RNA has also been reported and could
by the drug in the presence of a redox active
within the linker domain,
it has been suggested that
8
,9
10
or Cu. Metal independent
the methylvalerate moiety within the linker domain
assumes a specific compact conformation during com-
plexation with DNA. Methyl substitutions at positions
1
1
plausibly contribute to the therapeutic effects.
2and 4 also play important roles in stabilizing the
compact conformation essential for DNA binding.
2
4
BLM has four main structural domains (Fig. 1); the
function(s) of each have been studied by physical mea-
surements such as NMR, and by the structural modifi-
cation of individual domains. The metal binding domain
is responsible for metal binding, oxygen binding and
A
and Zn de-
also provided evidence for a bent con-
.
parallel study of the binding of Zn BLM
25,26
.
2
7,28
glycoBLM
formation of the DNA oligonucleotide-bound
metalloBLMs. In an effort to develop a conformationally
rigid deglycoBLM analogue that could mimic the beha-
vior of the parent molecule, and thereby provide evi-
dence in support of the putative bent structure of
(deglyco)BLM within the complex with DNA, we mod-
eled the conformation of deglycoBLM analogues con-
taining R-proline and S-proline in place of the
methylvalerate moiety normally present in deglycoBLM
3
,4
activation, and sequence selective cleavage of DNA.
The carbohydrate moiety participates in metal bind-
1
2,13
and possibly also in cell surface recognition.14 In
ing
the absence of the carbohydrate moiety the deglyco-
bleomycins exhibit a potency and sequence selective
1
5,16
DNA degradation similar to that of bleomycin.
The
(Fig. 2). These modeling studies suggested that the
deglycoBLM analogue containing S-proline (1a) was
reasonably similar in conformation to the conformation
*
7
Corresponding author. Tel.: +1-804-924-3906; fax: +1-804-924-
856; e-mail: sidhect@virginia.edu
0
968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.08.033

5180
A. Cagir et al. / Bioorg. Med. Chem. 11 (2003) 5179–5187
5 5
Figure 1. Structures and domains of BLM A and deglycoBLM A .
Figure 2. DeglycoBLM analogues containing S-proline (1a) and R-proline (1b) in place of the methylvalerate moiety.
.
calculated for Zn(II) deglycoBLM A (Fig. 3). Presently
the presence of Hunig’s base to afford resin 3. Mitsu-
5
5
3
we describe the syntheses and biochemical evaluation of
the deglycoBLM analogues containing S- and R-proline
in place of the methylvalerate moiety.
nobu reaction of resin 3 with N-Boc-3-aminopropan-
3
6
1-ol followed by Boc removal gave resin 4 (Scheme 1).
To quantify the loading on resin 4, a small amount of
the resin was treated with an excess of FmocOSu in the
3
7
presence of Hunig’s base. Quantitative Fmoc cleavage
indicated that the loading was 0.185 mmol/g, corre-
sponding to a yield of 81% for the three steps involved
in resin preparation. Resin 4 was used promptly for the
elaboration of deglycoBLMs 1.
Results
Syntheses of deglycoBLM analogues
The deglycoBLM analogues were prepared by solid
phase synthesis on a TentaGel resin in analogy with
Fmoc-protected bithiazole,2
the presence of HOBT, HBTU and Hunig’s base (Scheme
9ꢀ31
was coupled to resin 4 in
recent efforts from our laboratory.2
9ꢀ31
Resin 2 (Scheme
2ꢀ34
) was prepared as described by Bycroft3
loading 0.242 mmol/g. The terminal amine was pro-
tected with an o-nitrobenzenesulfonyl (NBS) group in
with a
2). This coupling proceeded in 88% yield, resulting in a
loading of 0.155 mmol/g. Fmoc deprotection (20%
piperidine in DMF), followed by condensation with
38
1

A. Cagir et al. / Bioorg. Med. Chem. 11 (2003) 5179–5187
5181
.
.
.
Figure 3. Comparison of Zn(II) deglycoBLM (green) with (A) Zn(II) deglycoBLM derivative 1a (red) and (B) Zn(II) deglycoBLM derivative 1b
red). The DNA binding domains (arrows) have been removed for clarity.
(
Scheme 1. Synthesis of mono-protected spermidine linked to a TentaGel resin.
Fmoc-threonine (HOBt, HBTU, and Hunig’s base) gave
the Fmoc-protected resin 5. The loading was found to
be 0.137 mmol/g for this step, corresponding to a 90%
yield.
The acid-labile protecting groups were removed suc-
cessfully with a 50% solution of 90:5:5 TFA–(i-
Pr) SiH–Me S in CH Cl over a period of 4 h. Removal
of the NBS group (1 N NaSPh in DMF) afforded the
resin-bound, fully deprotected deglycobleomycin analo-
gues 7a and 7b. Cleavage of these deglycobleomycins
from the resin was accomplished by three treatments of
each resin with 2% hydrazine in DMF. The filtrate was
concentrated under an argon atmosphere and the residue
3
2
2
2
The tripeptide linked resin 5 was divided into portions
to prepare the R- and S-proline-linked tetrapeptides.
Fmoc cleavage was achieved by treating resin 5 with
2
0% piperidine in DMF. The resin was coupled with
Fmoc-S-proline to produce the precursor of 1a, or
Fmoc-R-proline to produce the precursor of 1b
was dissolved in 0.5 mL of CF COOH. The deglyco-
3
BLM analogues precipitated following slow addition of
the CF COOH solution to ether cooled in dry ice. The
(
HATU, HOAT, and Hunig’s base). The loadings for
3
the tetrapeptide-linked resins were 0.132and 0.136 (97
and 100%), respectively, for the precursors of 1a and 1b.
After removal of the Fmoc group, both tetrapeptide-
linked resins were coupled with Fmoc-tritylhistidine
isolated solids were washed with 2mL of cold ether to
afford the crude products. DeglycoBLMs analogues 1a
and 1b were purified by C18 reversed-phase HPLC.
Purified deglycoBLMs 1a and 1b were collected and
lyophilized to yield the products as colorless solids.
(
HATU, HOAT, and Hunig’s base). Following Fmoc
1
deprotection, the loadings were found to be 0.129 mmol/
g for the precursor of 1a, and 0.124 mmol/g for the pre-
cursor of 1b (100 and 96%, respectively). The syntheses
These were characterized by ESI-MS and H NMR
spectroscopy.
of the fully protected deglycoBLM A analogues (6a and
5
Oxidative transformations mediated by deglycoBLMs 1
6
blamic acid
b) were completed by the addition of Boc-pyrimido-
4
0ꢀ42
to the resin-bound pentapeptides (excess
ꢁ
In previous studies, it has been reported that
.
Fe(III) BLM can function as a catalyst to transfer oxy-
gen to olefinic substrates in the presence of peroxide
BOP, and diisopropylethylamine at 0 C in the absence
of light under nitrogen) (Scheme 2).

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A. Cagir et al. / Bioorg. Med. Chem. 11 (2003) 5179–5187
Scheme 2. Synthesis and cleavage of resin-bound analogues to afford deglycoBLMS 1a and 1b.
(
Scheme 3).43,44 Accordingly, deglycoBLM analogues 1a
hyde, albeit with somewhat lower efficiency than that of
bleomycin itself.
3
+
and 1b were complexed with Fe and used as catalysts
to oxidize styrene in the presence of excess hydrogen
peroxide. As shown in Table 1, both of these con-
formationally constrained analogues were capable of
oxidizing styrene to styrene oxide and phenylacetalde-
The cleavage of DNA by deglycoBLM analogues 1a
and 1b was studied initially using supercoiled pSP64
plasmid DNA as a substrate. As shown in Figure 4, the
use of increasing concentrations of 1a or 1b in the pre-
2
+
sence of 1.5 mM Fe resulted in increasing conversion
of Form I (supercoiled) to Form II (nicked circular)
DNA. Unlike deglycoBLM A (lanes 13 and 14), neither
5
1a nor 1b produced any detectable Form III (linear
duplex) DNA. The conversion of Form I to Form II
DNA was comparable for deglycoBLM 1a than 1b (cf.
Scheme 3. Oxidation of styrene by deglycobleomycin A
a and 1b.
5
derivatives
1
Table 1. Oxygen transfer to styrene mediated by bleomycin and conformationally constrained BLM analogues 1a and 1b in the presence of Fe3+a
BLM
derivative
Oxidized product
Product conc (mM)
80 min
4
0 min
120 min
Bleomycinb
Phenylacetaldehyde
Styrene oxide
Phenylacetaldehyde
Styrene oxide
Phenylacetaldehyde
Styrene oxide
2.11ꢂ0.61
4.56ꢂ2.84
0.64ꢂ0.09
0.53ꢂ0.04
ꢂ0.04
5.31ꢂ2.21
0.51ꢂ0.13
0.70ꢂ0.40
0.57ꢂ0.27
0.38ꢂ0.04
0.38ꢂ0.05
0.78ꢂ0.04
DeglycoBLM 1ac
DeglycoBLM 1bd
0.56ꢂ0.22
0.68ꢂ0.420.52
0.37ꢂ0.020.38
0.44ꢂ0.04
ꢂ0.03
0.40ꢂ0.05
a
All concentrations are in mM, and all values are the mean of three experiments.
Final concentration in reaction mixture was 0.79 mM.
Final concentration in reaction mixture was 0.49 mM.
b
c
d
Final concentration in reaction mixture was 0.35 mM.

A. Cagir et al. / Bioorg. Med. Chem. 11 (2003) 5179–5187
5183
sequence selective DNA cleavage.3 Structurally mod-
ified BLM analogues have been used to define the roles
of the metal binding domain in DNA recognition. These
studies have established that the metal binding domain
ꢀ5
4
5
46
can determine both sequence and strand selectivity
of DNA cleavage. While the metal binding domain of
deglycobleomycin alone was not able to cleave DNA in
4
7
a sequence selective fashion,
presumably due to
inadequate affinity for DNA, a structural analogue of
this domain was shown to mediate DNA cleavage with
a selectivity quite similar to that of BLM itself.4
8
Figure 4. Cleavage of supercoiled pSP64 DNA by deglycoBLM ana-
logues 1a and 1b. Lane 1: DNA alone; lane 2: 1.5 mM Fe ; lane 3: 5
2+
While the metal binding domain of BLM is believed to
be the primary determinant of sequence selective clea-
vage by BLM, studies involving photoinduced DNA
cleavage by halogenated bithiazoles indicated that these
species can bind to DNA in a sequence selective fashion,
2+
mM 1b; lane 4: 1 mM 1b+1.5 mM Fe ; lane 5: 3 mM 1b+1.5 mM
2
+
2+
Fe ; lane 6: 5 mM 1b+1.5 mM Fe ; lane 7: 5 mM 1a; lane 8: 1 mM
1
2+
2+
a+1.5 mM Fe ; lane 9: 3 mM 1a+1.5 mM Fe ; lane 10: 5 mM
2+
1
a+1.5 mM Fe ; lane 11: 5 mM deglycoBLM A
5
; lane 12: 1 mM
+1.5 mM
.
2+
dgBLM A5+1.5 mM Fe ; lane 13: 3 mM deglycoBLM A
5
2+
2+
49,50
Fe ; lane 14: 5 mM deglycoBLM A
5
+1.5 mM Fe
albeit with a selectivity different than that of BLM.
When the chlorinated bithiazoles were incorporated
within deglycoBLM analogues, these species mediated
photoactivated DNA cleavage in the same fashion as
the chlorinated bithiazoles. Alternatively, cleavage of
the same DNA substrate could be effected in dark reac-
2
+
tions by admixture of Fe
Oxidative cleavage exhibited sequence selectivity of
under aerobic conditions.
5
1
DNA cleavage characteristic of (deglyco)BLM itself.
In the aggregate, these findings suggested that both the
metal binding and C-terminal domains of (deglyco)-
BLM mediate sequence selective DNA binding. It seems
likely that DNA cleavage is maximally efficient at sites
that can accommodate the binding preferences of both
of the foregoing structural domains of BLM. In this
context, one would expect that the linker region that
connects the metal binding and C-terminal domains
should be able to facilitate the binding of the attached
domains to DNA. In this sense, it is not surprising that
1
8ꢀ21,25ꢀ28
both spectroscopic
and synthetic studies have
suggested a compact folded structure for the linker
domain of (deglyco)BLM.
Figure 5. Cleavage of a 5 -32P end labeled DNA duplex by deglyco-
0
BLM analogues 1a and 1b. Lane 1: DNA alone; lane 2: 1 mM 1b+10
2+
2+
In the present study, two analogues of deglyco(BLM)
have been prepared in which proline has replaced the
methylvalerate moiety normally present. The analogue
containing S-proline (1a) was predicted to have a con-
formation reasonably similar to that calculated for
Zn(II) deglycoBLM (Fig. 3). In comparison, analogue
1b containing R-proline was predicted to assume a
rather different conformation.
mM Fe ; lane 3: 10 mM 1b+10 mM Fe ; lane 4: 1 mM 1a+10 mM
2
+
2+
Fe ; lane 5: 10 mM 1a+10 mM Fe ; lane 6: 1 mM deglycoBLM
2+
2+
5 5
A +10 mM Fe ; lane 7: 10 mM deglycoBLM A +10 mM Fe . The
band of intermediate mobility in lanes 1–5 was due to adventitious
non-denatured DNA.
.
lanes 6 and 10), but not the result that might have been
anticipated based on initial modeling studies (Fig. 3).
0
32
.
Fe(II) deglycoBLMs 1a and 1b were both found to
Also studied was the cleavage of a 5 - P end labeled
.
158-base pair DNA duplex by Fe(II) deglycoBLMs 1a
and 1b. As shown in Figure 5, both analogues effected
significant DNA cleavage when employed at 10 mM
concentration in the presence of equimolar Fe , but
neither produced sequence selective cleavage of the
DNA duplex substrate.
transfer oxygen to styrene, verifying that both could
activate oxygen for DNA cleavage (Table 1). While
both analogues mediated concentration dependent
relaxation of supercoiled plasmid DNA (Fig. 4), as well
as the degradation of linear duplex DNA (Fig. 5), nei-
ther deglycoBLM analogue effected DNA cleavage in a
sequence selective fashion.
2
+
In principle, the lack of sequence selective DNA clea-
vage may simply reflect the inadequacy of con-
formationally constrained proline analogues 1a or 1b as
models of the DNA-bound conformation of (deglyco)-
Discussion
Several studies have been reported that analyze the roles
of the individual structural domains of BLM in

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A. Cagir et al. / Bioorg. Med. Chem. 11 (2003) 5179–5187
.
27
BLM defined for Zn(II) deglycoBLM. In this context,
it is interesting to note that a parallel study has utilized
an analysis of the folded conformation of the methyl-
acid, followed by five washings with 2-mL portions of
DMF, five washings with 2-mL portions of CH Cl , and
2
2
two washings with 2-mL portions of DMF. The resin
was neutralized by washing five times with 2-mL por-
tions of a 0.1 N solution of diisopropylethylamine in
DMF and then five times with 2-mL portions of DMF,
five times with 2-mL portions of CH Cl , and two times
.
18-24
valerate moiety of Co(III) BLM
to synthesize
another series of deglycoBLM analogues containing
conformationally constrained analogues designed to
mimic elements of that structural motif. Those analo-
gues also failed to mediate sequence selective DNA
cleavage.
5
2
2
2
with 2-mL portions of DMF to yield resin 4. A small
amount of resin 4 in 0.5 mL of DMF was treated with
an excess (10 equiv) of FmocCl and ꢃ30 equiv of
Hunig’s base; the mixture was shaken for 30 min. The
resin was filtered and rinsed with five 2-mL portions of
DMF, five 2-mL portions of CH Cl , and two 2-mL
While the failure of each of the prepared analogues to
mediate DNA cleavage in a selective fashion may only
reflect the inability of the analogues to assume a precise
conformation required for DNA cleavage, it seems pos-
sible that the intrinsic lack of flexibility of the con-
formationally constrained analogues could also
contribute to the lack of sequence selective DNA clea-
vage. If the latter were true, it would suggest that an
initially formed metalloBLM complex must undergo a
conformational rearrangement before DNA cleavage
can occur. While no direct test of this possibility has
2
2
portions of MeOH, then dried under vacuum. The
loading was determined from the Fmoc cleavage assay
as 0.185 mmol/g (81% yield).
To 500 mg (0.121 mmol) of resin 4 was added a solution
2
9-31
containing 173 mg (0.36 mmol) of Fmoc bithiazole,
138 mg (0.36 mmol) of HBTU (O-benzotriazolyl-
0
0
N,N,N ,N -tetramethyluronium hexafluorophosphate),
49 mg (0.36 mmol) of HOBT (N-hydroxybenzotriazole),
and 126 mL (93 mg, 0.37 mmol) of Hunig’s base in 2
mL of DMF. The resulting mixture was shaken for 30
min. The resin was filtered and rinsed with five 2-mL
portions of DMF, five 2-mL portions of CH Cl , and
.
been made, it has been reported that Co(III) BLM binds
5
3
to sites on DNA that it does not cleave and that a
.
conformational rearrangement of Co(III) BLM bound
to DNA may be required for double-stranded DNA
4ꢀ56
5
cleavage.
2
2
two 2-mL portions of MeOH, then dried under vacuum
over KOH pellets. A small sample was subjected to the
Kaiser test, which indicated that the reaction was com-
plete; quantitative Fmoc cleavage indicated a loading of
0.155 mmol/g (88% yield).
Experimental
Synthesis of deglycoBLM analogues
Protected resin-bound deglycoBLMs 6a and 6b. To 600
mg (0.27 mmol) of pre-swollen resin 2 was added 239
mg (1.08 mmol) of o-nitrobenzenesulfonyl chloride and
A solution containing 2mL of 20 % piperidine in DMF
was added to the pre-swollen resin-linked dipeptide. The
suspension was shaken for 5 min, filtered and treated
again with 2mL of 02 % piperidine in DMF for an
additional 5 min. This rinsing/shaking procedure was
repeated twice. The resin was washed with five 2-mL
portions of DMF, five 2-mL portions of CH Cl , and
two 2-mL portions of DMF. A solution containing 79
mg (0.23 mmol) of Fmoc threonine, 88 mg (0.23 mmol)
of HBTU, 31 mg (0.23 mmol) of HOBT, and 81 mL (60
mg, 0.47 mmol) of Hunig’s base in 2mL of DMF was
added to the resulting resin, which was shaken gently.
After 30 min resin 5 was filtered and rinsed with five
2
2
81 mL (208 mg, 1.62 mmol) of diisopropylethylamine in
mL of dry THF. The reaction mixture was shaken in
the absence of light under nitrogen for 5 h to obtain
resin 3. This resin was filtered and washed five times
with 2-mL portions of dry THF, followed by five wash-
ings with 2-mL portions of DMF, and two with 2-mL
portions of CH Cl . A small amount of resin was sub-
2
2
2
2
jected to the Kaiser test to verify completion of the
coupling reaction. The resin was used in the next step
immediately.
2-mL portions of DMF, five 2-mL portions of CH Cl ,
2 2
To 600 mg (0.27 mmol) of pre-swollen resin was added
slowly 439 mg (1.62mmol) of N-Boc-3-aminopropanol,
and 364 mg (1.35 mmol) of triphenylphosphine in a
and two 2-mL portions of DMF. Quantitative Fmoc
cleavage indicated a loading of 0.137 mmol/g (90%
yield).
2
5-mL column fitted with a frit. The resin was shaken
gently for 1 min, then 0.33 mL (365 mg, 2.0 mmol) of
diethyl azodicarboxylate was added slowly. The resin
was shaken for 24 h, then filtered and washed five times
with 2-mL portions of DMF, followed by five 2-mL
portions of CH Cl to afford Nbs- and Boc-protected
A solution containing 2mL of 20 % piperidine in DMF
was added to 276 mg (0.038 mmol) of pre-swollen resin
5 in a 6-mL column fitted with a frit. The suspension
was shaken for 5 min, filtered and again treated with 2
mL of 20% piperidine in DMF for 5 min. This rinsing/
shaking procedure was repeated twice. The resin was
washed with five 2-mL portions of DMF, five 2-mL
portions of CH Cl , and two 2-mL portions of DMF. A
2
2
spermidine resin.
To a suspension of 500 mg (0.121 mmol) of swollen,
fully protected spermidine resin was added 1 mL of
2
2
solution containing 38 mg (0.114 mmol) of Fmoc-S-
proline (for 1a) or Fmoc-R-proline (for 1b), 43 mg
(0.114 mmol) of HATU (O-(7-azabenzotriazol-1-yl)-
0
.1 M triisopropylsilane in CH Cl . Then 1.2mL (1.78
2 2
g, 16 mmol) of trifluoroacetic acid was added and the
resin was shaken for 2h. The resin was filtered and
washed twice with 1.5-mL portions of trifluoroacetic
0
0
N,N,N ,N -tetramethyluronium hexafluorophosphate),
16 mg (0.114 mmol) of HOAT (1-hydroxy-7-azabenzo-

A. Cagir et al. / Bioorg. Med. Chem. 11 (2003) 5179–5187
5185
triazole), and 40 mL (30 mg, 0.37 mmol) of Hunig’s base
in 0.5 mL of DMF was added. After 30 min, the resin
was filtered and rinsed with five 2-mL portions of DMF,
five 2-mL portions of CH Cl , and two 2-mL portions
of MeOH, and then dried under vacuum. Quantitative
Fmoc cleavage indicated a loading of 0.132mmol/g
(97% yield) for S-proline, and 0.136 mmol/g (100%
yield) for R-proline attachment to the resin-bound
peptide.
phenolate solution and washing, deglycoBLMs 1a and
1b were cleaved from the resin by three treatments with
0.5 mL of 2% hydrazine in DMF for 3 min. The filtrates
were collected in 5-mL round-bottom flasks. The solu-
tions were concentrated under an argon atmosphere.
The products were dissolved in 0.5 mL of trifluoroacetic
2
2
ꢁ
acid and precipitated in cold anhydrous ether (ꢀ20 C)
as colorless solids. The precipitates were washed with 2
mL of cold anhydrous ether and dried under diminished
pressure. The solids were dissolved in 1 mL of deionized
water, frozen and lyophilized to give colorless solids.
Chromatographic purification was performed on an
Alltech Alltima C18 reversed-phase HPLC column
(150Â4.6 mm) using a gradient of aq 0.1 N trifluoro-
acetic acid and acetonitrile. A linear gradient was
employed (88:120.1 N CF COOH–CH CN!79:21
A solution containing 2mL of 20 % piperidine in DMF
was added to the pre-swollen resin containing tetrapep-
tide (0.038 mmol). The suspension was shaken for 5
min, filtered and treated again with 2mL of 02 %
piperidine in DMF for an additional 5 min. This rin-
sing/shaking procedure was repeated twice. The resin
was washed with five 2-mL portions of DMF, five 2-mL
portions of CH Cl , and two 2-mL portions of DMF. A
3
3
0.1 N CF COOH–CH CN over a period of 30 min at a
3
3
flow rate of 4 mL/min). Fractions containing the desired
product were collected, frozen and lyophilized to afford
a colorless solid: yield 1.1 mg of 1a, and 1.8 mg of 1b.
2
2
solution containing 71 mg (114 mmol) of Fmoc tri-
tylhistidine, 43 mg (0.114 mmol) of HATU, 16 mg
(
0.114 mmol) of HOAT, and 40 mL (30 mg, 0.37 mmol)
1
of Hunig’s base in 0.5 mL of DMF was added. After 30
min, the resin was filtered and rinsed with five 2-mL
portions of DMF, five 2-mL portions of CH Cl , and
two 2-mL portions of MeOH. The resulting resin was
dried under diminished pressure over KOH pellets.
Quantitative Fmoc removal indicated a loading of 0.129
mmol/g (100% yield) for the S-proline derivative, and
For 1a: H NMR (D O) d 1.05 (d, 3H, J=6.4 Hz),
2
1.57–1.77 (m, 5H), 1.80–1.95 (m, 7H), 2.10 (m, 1H),
2.69–2.81 (m, 1H), 2.90–3.20 (m, 13H), 3.45–3.70 (m,
6H), 3.80 (m, 1H), 4.00–4.20 (m, 4H), 4.40 (m, 1H), 5.10
(m, 1H), 7.25 (s, 1H), 8.03 (s, 1H), 8.12 (s, 1H) and 8.56
2
2
+
(s, 1H); mass spectrum (ESI), m/z 1025.5 (M+H) ,
+
+
+++
513.4 (M+H)
+
, and 343.0 (M+H)
(theoretical
0
.124 mmol/g (96% yield) for R-proline derivative.
(M+H) 1026.2).
1
A solution containing 2mL of 20 % piperidine in DMF
was added to 46 mg (0.006 mmol) of pre-swollen resin
containing the pentapeptide in a 6-mL column fitted
with a frit. The suspension was shaken for 5 min, fil-
tered and again treated with 2mL of 20 % piperidine in
DMF for 5 min. This rinsing/shaking procedure was
repeated twice. The resin was washed with five 2-mL
portions of DMF, five 2-mL portions of CH Cl , and
For 1b: H NMR (D O) d 1.00 (d, 3H, J=6.4 Hz),
2
1.59–1.77 (m, 4H), 1.77–2.00 (m, 8H), 2.20 (m, 1H),
2.59–2.67 (m, 1H), 2.85–3.31 (m, 13H), 3.39–3.64 (m,
6H), 3.80 (m, 1H), 3.95–4.20 (m, 4H), 4.42 (m, 1H), 5.14
(m, 1H), 7.26 (s, 1H), 7.95 (s, 1H), 8.12 (s, 1H) and 8.56
+
(s, 1H); mass spectrum (ESI), m/z 1025.6 (M+H) ,
+
+
+++
513.6 (M+H)
+
, and 342.9 (M+H)
(theoretical
(M+H) 1026.2).
2
2
two 2-mL portions of DMF. A solution containing 7.5
4
0ꢀ42
24
mg (0.018 mmol) of N-Boc pyrimidoblamic acid,
mg (0.053 mmol) of BOP (benzotriazol-1-yl-oxy-tris–
dimethylaminophosphonium hexafluorophosphate),
General procedure for the oxidation of styrene by
.
Fe(III) BLM and analogues
and 18 mL (13 mg, 0.11 mmol) of Hunig’s base in 0.3
mL of DMF was added, and protected from light. After
To a solution of 0.20 mg (0.19 mmol) of BLM deriva-
tive, and 0.09 mg (0.19 mmol) of ferric perchlorate in 220
mL of water was added at 4 C 100 mL of a methanolic
ꢁ
16 h the resin was filtered and rinsed with five 2-mL
portions of DMF and five 2-mL portions of CH Cl to
yield resins 6a and 6b.
solution containing 277 mM styrene and 6.21 mM ethyl
benzoate. To the resulting solution was added a 91 mM
H O solution until a final concentration of 15 mM
2
2
2
2
H O was reached. Aliquots (100-mL) of the reaction
mixture were taken at 40-min time intervals, and each
was quenched with 300 mL of water, and extracted
2
2
Deprotection and resin cleavage of deglycoBLM analogues
To the suspension of fully protected resin-linked degly-
cobleomycins 6a and 6b was added 1 mL of 20:1:1
CH Cl –(i-Pr) SiH–Me S. Then 1.2mL (19.2mmol) of
with three 100-mL portions of CHCl . The combined
3
organic phase was dried over MgSO , filtered and
2
2
3
2
4
trifluoroacetic acid was added and the resin was shaken
for 2h. The resin was filtered and washed twice with
analyzed by gas chromatography. Injections were 10 mL
in volume (splitless mode) and applied to a 5% phenyl-
siloxane capillary column (0.25 mm IDÂ30 m). The
following temperature program was employed at a He
1
2
.5-mL portions of trifluoroacetic acid, followed by five
-mL portions of DMF, five 2-mL portions of CH Cl ,
2
2
ꢁ
and two 2-mL portions of DMF. The resin was treated
with 2mL of 1 N sodium thiophenolate in DMF for 20
min to obtain resins 7a and 7b. The resins were filtered
and washed with five 2-mL portions of DMF, five 2-mL
portions of CH Cl , and two 2-mL portions of DMF.
gas flow rate of 1.0 mL/min: 60 C for 22 min;
ꢁ
ꢁ
ꢁ
60!85 C at 10 C/min; then 85 C for 15 min. Under
these conditions the observed retention times (cor-
rected) were as follows: styrene 4.6 min; phenylacet-
aldehyde 18.9 min; styrene oxide 21.3 min; ethyl
benzoate 31.0 min.
2
2
After an additional treatment with 0.1 N sodium thio-

5
186
A. Cagir et al. / Bioorg. Med. Chem. 11 (2003) 5179–5187
13. Akkerman, M. A. J.; Neijman, E. W. J. F.; Wijmenga,
S. S.; Hilbers, C. W.; Bermel, W. J. Am. Chem. Soc. 1990, 112,
Molecular modeling studies
.
7462.
Modeling studies of the Zn(II) deglycoBLM and the R-
and S-proline analogues were carried out using Insight
II/Discover_3 (Accelrys, San Diego, CA, USA) on a
Silicon Graphics Octane using the ESFF forcefield. The
screw sense, axial ligand and bond angles around the Zn
metal were fixed based on the results of previous mod-
1
2
1
4. Choudhury, A. K.; Tao, Z.-F.; Hecht, S. M. Org. Lett.
001, 3, 1291.
5. Aoyagi, Y.; Suguna, H.; Murugesan, N.; Ehrenfeld, G. M.;
Chang, L.-H.; Ohgi, T.; Shekhani, M. S.; Kirkup, M. P.;
Hecht, S. M. J. Am. Chem. Soc. 1982, 104, 5237.
16. Sugiyama, H.; Ehrenfeld, G. M.; Shipley, J. P.; Kilk-
uskie, R. E.; Chang, L.-H.; Hecht, S. M. J. Nat. Prod. 1985,
48, 869.
17. Chien, M.; Grollman, A. P.; Horwitz, S. B. Biochemistry
1977, 16, 3641.
18. Wu, W.; Vanderwall, D. E.; Lui, S. M.; Tang, X.-J.;
Turner, C. J.; Kozarich, J. W.; Stubbe, J. J. Am. Chem. Soc.
1
8,27
eling studies.
Molecular dynamics followed by mini-
.
mization of Zn(II) deglycoBLM and the respective R-
and S- proline analogues produced significantly different
results (Fig. 3). Ten structures were calculated for each
analogue. The majority of structures fell within a narrow
range for each set of structures. We observed a high
degree of structural similarity in the metal binding
domain and the methylvalerate and proline peptides,
respectively. The threonine and bithiazole regions
showed progressively higher variability moving away
from the metal binding domain. Similar to the reported
1
1
996, 118, 1268.
9. Wu, W.; Vanderwall, D. E.; Turner, C. J.; Kozarich, J. W.;
Stubbe, J. J. Am. Chem. Soc. 1996, 118, 1281.
0. Lui, S. M.; Vanderwall, D. E.; Wu, W.; Tang, X.-J.;
Turner, C. J.; Kozarich, J. W.; Stubbe, J. J. Am. Chem. Soc.
997, 199, 9603.
2
1
.
.
18
structure of HOO Co(III) BLM
the bithiazole of
21. Wu, W.; Vanderwall, D. E.; Teramoto, S.; Lui, S. M.;
Hoehn, S.; Tang, X.-J.; Turner, C. J.; Boger, D. L.; Kozarich,
J. W.; Stubbe, J. J. Am. Chem. Soc. 1998, 120, 2239.
,
.
Zn(II) deglycoBLM was folded back underneath the
equatorial plane of the metal binding domain and was
on the opposite face relative to the axial primary amine
ligand. The metal binding domains of the R- and S-
proline analogues were superimposed on the metal
2
2. Boger, D. L.; Colletti, S. L.; Teramoto, S.; Ramsey, T. M.;
Zhou, J. Bioorg. Med. Chem. 1995, 3, 1281.
3. Boger, D. L.; Ramsey, T. M.; Cai, H.; Hoehn, S. T.;
Stubbe, J. J. Am. Chem. Soc. 1998, 120, 9139.
4. Boger, D. L.; Ramsey, T. M.; Cai, H.; Hoehn, S. T.;
Stubbe, J. J. Am. Chem. Soc. 1998, 120, 9149.
5. Manderville, R. A.; Ellena, J. F.; Hecht, S. M. J. Am.
2
.
binding domain of Zn(II) deglycoBLM to visualize the
similarity of methylvalerate and proline congeners.
2
2
Chem. Soc. 1994, 116, 10851.
26. Manderville, R. A.; Ellena, J. F.; Hecht, S. M. J. Am.
Chem. Soc. 1995, 117, 7891.
Acknowledgements
We thank Dr. Christopher Leitheiser for advice on the
solid phase synthesis of the deglycoBLM analogues.
This work was supported by NIH Research Grants
CA76297 and CA77284, awarded by the National Can-
cer Institute.
27. Sucheck, S. J.; Ellena, J. F.; Hecht, S. M. J. Am. Chem.
Soc. 1998, 120, 7450.
2
2
8. Sucheck, S. J. PhD Thesis, University of Virginia, 1998.
9. Leitheiser, C. J.; Rishel, M. J.; Wu, X.; Hecht, S. M. Org.
Lett. 2000, 2, 3397.
0. Smith, K. L.; Tao, Z.-F.; Hashimoto, S.; Leitheiser, C. J.;
Wu, X.; Hecht, S. M. Org. Lett. 2002, 4, 1079.
1. Tao, Z.-F.; Leitheiser, C. J.; Smith, K. L.; Hashimoto, S.;
Hecht, S. M. Bioconjugate Chem. 2002, 13, 426.
2. Bycroft, B. W.; Chan, N. D.; Hone, S.; Millington, S.;
3
3
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3
7
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4